JP2014514540A - 感染の早期診断およびモニタリングのための白血球の自己蛍光を使用した細胞学的方法 - Google Patents
感染の早期診断およびモニタリングのための白血球の自己蛍光を使用した細胞学的方法 Download PDFInfo
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Abstract
Description
重度敗血症または重篤な細菌感染は、院内、より具体的には、集中治療室における罹患率、死亡率の主因であり続けている。さらに、米国民1,000人に3〜11例という現在の罹患率は、過去数十年にわたり毎年8.7%ずつ増加してきた。フランスにおける集中治療室への入院の14.6%が、この病理に関係している。診断および治療の手法の多くの改善にも関わらず、重度敗血症に関連した院内死亡率は、フランスにおいて、35%と推定されている。患者の入院から、抗生物質による治療の開始までの期間が、死亡率の主要な予後因子を構成することは、現在、明らかである。従って、21世紀における目標は、可能な限り早期に感染を診断することであり、それは、患者の生存の保証である。
(i)該試料の平均細胞自己蛍光強度を測定する工程、および
(ii)該個体の感染状態を判定するため、工程(i)において測定された強度を対照値と比較する工程。
(a)試料の細胞を、少なくとも1種の細胞遠心分離(cytocentrifugation)システムに投入する工程、
(b)細胞をスライド上に塗抹するため、工程(a)で入手した装置を、好ましくは約600rpmで、約5分間、遠心分離する工程、
(c)細胞スポットを形成させるため、スライド上に塗抹された細胞を、少なくとも1滴の約4%パラホルムアルデヒド(PFA)により、好ましくは約10分間、固定する工程、
(d)予め固定した細胞スポットを、好ましくはPBS緩衝液により、濯ぐ工程、
(e)予め濯いだ細胞スポットを乾燥させる工程、
(f)予め乾燥させた細胞スポットに、蛍光の観察に適合する封入剤を少なくとも1滴、添加する工程、
(g)工程(f)から得られた細胞スポットに、好ましくはガラス製の、透明カバーガラスを載せる工程。
本発明の方法は、リスクを有する個体における細菌性および/またはウイルス性の病原体の存在を診断するため、免疫系の特別な細胞の自己蛍光を使用する。細菌性および/またはウイルス性の感染性病原体は、TLRの活性化を介して、これらの免疫細胞における初期代謝活性を引き起こすことができる。この代謝活性は、その細胞内自己蛍光を特徴とする、細胞エネルギー代謝に関与している必須の補酵素のうちの1種であるNAD(P)Hの濃度の変動と相関し得る(Mayevsky A et al.,Am J.Physiol.Cell Physiol.2007)。
(i)該試料の平均細胞自己蛍光強度を測定する工程、および
(ii)該個体の感染状態を判定するため、工程(i)において測定された強度を対照値と比較する工程。
(a)試料からの細胞を、少なくとも1種の細胞遠心分離システムに投入する工程、
(b)細胞をスライド上に塗抹するため、好ましくは約600rpmで、約5分間、工程(a)で入手した装置を遠心分離する工程、
(c)細胞スポットを形成させるため、スライド上に塗抹された細胞を、少なくとも1滴の約4%パラホルムアルデヒド(PFA)により、好ましくは約10分間、化学的に固定する工程、
(d)好ましくはPBS緩衝液により、予め固定した細胞スポットを濯ぐ工程、
(e)予め濯いだ細胞スポットを乾燥させる工程、
(f)予め乾燥させた細胞スポットに、蛍光の観察に適合する封入剤を少なくとも1滴、添加する工程、
(g)工程(f)から得られた細胞スポットに、好ましくはガラス製の、透明カバーガラスを載せる工程。
(a)細胞を、少なくとも1種の細胞遠心分離システムに投入する工程、
(b)細胞をスライド上に塗抹するため、工程(a)で入手した装置を、好ましくは約600rpm(41g)で、約5分間、遠心分離する工程、
(c)細胞スポットを形成させるため、スライド上に塗抹された細胞を、少なくとも1滴の約4%パラホルムアルデヒド(PFA)により、好ましくは約10分間、化学的に固定する工程、
(d)予め固定した細胞スポットを、好ましくはPBS緩衝液により、濯ぐ工程、
(e)予め濯いだ細胞スポットを乾燥させる工程、
(f)予め乾燥させた細胞スポットに、蛍光の観察に適合する封入剤を少なくとも1滴、添加する工程、
(g)工程(f)から得られた細胞スポットに、好ましくはガラス製の、透明カバーガラスを載せる工程。
(1)患者に由来する試料の自動調製、
(2)試料の平均細胞自己蛍光の自動測定、
(3)値の対照値との比較、
(4)結果の生成:患者における細菌性、ウイルス性、または真菌性の病原体の存在。
全血を、3.8%(w/v)クエン酸ナトリウム0.5mlを含む4.5ml BD Vacutainer(登録商標)チューブに採取した。培養培地RPMI 1640は、Invitrogen(登録商標)(Paisley,United Kingdom)製である。
初期の細胞観察に基づき、NAD(P)H自己蛍光強度の画像分解能および生物学的試料の悪化に関して最良の折衷を与える最適の光学実験条件を設定した。
有志者のインフォームドコンセントの後、8人の健康有志者から20mlの全血を採取した。健康有志者の集団は、少なくとも7日間、いかなる処置も受けていない、特に、抗生物質および抗炎症薬を受けていない、25〜60才の男女から構成されていた。彼らは、明らかに発生中の感染を示さなかった。血液は、肘の内側の静脈の穿刺によって入手した。
当業者に周知の密度勾配技術(Lympholyte-Poly(登録商標)(Cedarlane Tebu-bio(登録商標),Le Perrey-en-Yvelines,France))を使用して、各血液試料(20ml)から、単核細胞(MNC)を単離した。この技術は、2本の別個の細胞のバンド、回収されたMNC(リンパ球および単球)の上方バンドと、多形核好中球から本質的に構成される多核細胞の下方バンドとを入手することを可能にする。次いで、磁気カラム(MACS(登録商標)(Miltenyi Biotec,Paris))での分離によるポジティブ選択技術を使用して、MNCから単球を単離した。次いで、血中単球を、RPMI 1640+4%ヒト血清+ペニシリンG(100U/ml)およびストレプトマイシン(100ng/ml)(Valbiotech)に浮遊させた。
各血液試料(12ml)から、Ficolでの遠心分離(40分、1600rpm、+4℃)により、RPMI+ヒト血清(4%)+EDTA(5%)に浮遊した下方バンドを回収することができた。次いで、この培地で2回の洗浄を実施した(10分、1600rpm、+4℃)。次いで、磁気カラム(MACS(登録商標)(Miltenyi Biotec,Paris))での分離によるネガティブ選択により、抗CD16抗体の添加による好酸球の枯渇を実施した。次いで、PNNを、RPMI 1640+4%ヒト血清+ペニシリンG(100U/ml)およびストレプトマイシン(100μg/ml)(Valbiotech)に浮遊させた。
ブドウ球菌肺炎のモデルに関しては、メチシリン感受性黄色ブドウ球菌(MSSA)の株(ATCC 29213株)を、トリプティックソイ(tryptic soy)培地において37℃で16時間増殖させた。使用直前に、培養物を2回洗浄し(1000gで10分間遠心分離し)、無菌の等張生理食塩水で希釈し、それを分光法によって較正した。細菌濃度を、定量培養によって系統的にモニタリングした。
Claims (12)
- 少なくとも以下の工程を含む、個体に由来する生物学的標本から得られた白血球細胞の試料に基づき、該個体の感染状態を診断するためのインビトロの方法:
(i)該試料の平均細胞自己蛍光強度を測定する工程、および
(ii)該個体の感染状態を判定するため、工程(i)において測定された強度を対照値と比較する工程。 - 試料の白血球細胞が、単球および/または多形核好中球より選択されることを特徴とする、請求項1記載の方法。
- 個体の状態が感染性である場合、該個体が、細菌感染、ウイルス感染、または真菌感染、好ましくは細菌感染を患っていることを特徴とする、請求項1または2記載の方法。
- 生物学的試料が、個体の器官から得られた流体であることを特徴とする、請求項1〜3のいずれか一項記載の方法。
- 生物学的試料が、肺液(pulmonary fluid)、腹水、脳脊髄液、血液試料、またはその他の任意の感染している可能性のある器官の液体であることを特徴とする、請求項1〜4のいずれか一項記載の方法。
- 試料の細胞の自己蛍光強度が、浮遊細胞において測定されることを特徴とする、請求項1〜5のいずれか一項記載の方法。
- 浮遊細胞の自己蛍光強度が、フローサイトメトリーまたは分光測光によって測定されることを特徴とする、請求項6記載の方法。
- 試料の細胞の自己蛍光強度が、蛍光の観察に適合するスライド、好ましくは透明スライドガラスに化学的に固定され配置された細胞において測定されることを特徴とする、請求項1〜5のいずれか一項記載の方法。
- 試料の細胞の自己蛍光強度が、蛍光顕微鏡を使用して測定されることを特徴とする、請求項1〜8のいずれか一項記載の方法。
- 細胞の試料が、少なくとも以下の工程を含む方法により、工程(i)の前に、透明材料のスライド上に単層で調製される、請求項1〜5、8、および9のいずれか一項記載の方法:
(a)試料の細胞を、少なくとも1種の細胞遠心分離(cytocentrifugation)システムに投入する工程、
(b)細胞をスライド上に塗抹するため、工程(a)で入手した装置を、好ましくは約600rpmで、約5分間、遠心分離する工程、
(c)細胞スポットを形成させるため、スライド上に塗抹された細胞を、少なくとも1滴の約4%パラホルムアルデヒド(PFA)により、好ましくは約10分間、化学的に固定する工程、
(d)予め固定した細胞スポットを、好ましくはPBS緩衝液により、濯ぐ工程、
(e)予め濯いだ細胞スポットを乾燥させる工程、
(f)予め乾燥させた細胞スポットに、蛍光の観察に適合する封入剤を少なくとも1滴、添加する工程、
(g)工程(f)から得られた細胞スポットに、好ましくはガラス製の、透明カバーガラスを載せる工程。 - 試料の白血球細胞が主として単球である場合、工程(i)において測定された自己蛍光強度が対照値より有意に高いならば個体の状態が感染性であることを特徴とする、前記請求項のいずれか一項記載の方法。
- 試料の白血球細胞が主として多形核好中球である場合、工程(i)において測定された自己蛍光強度が対照値より有意に低いならば個体の状態が感染性であることを特徴とする、前記請求項のいずれか一項記載の方法。
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